Monoclonal antibodies to porcine factor VIII coagulant and their use in the isolation of active coagulant protein.
Partially purified preparations of porcine factor VIII:C were used to immunize mice and spleen cells from the immunized animals were fused to NS-1 mouse myeloma cells. The ability of hybrid culture fluids to bind factor VIII:C was detected with a radiolabelled, affinity-purified, human antihuman VIII:C inhibitor. Three cloned hybrid lines have been obtained that preferentially bind to VIII:C when compared to von Willebrand factor binding. Two of these monoclonal antibodies partially inhibit VIII:C coagulant activity. The third antibody does not inhibit VIII:C, but it can be used as an affinity reagent to absorb dissociated VIII:C out of solution. Active coagulant can be recovered by elution in 50% ethylene glycol. The VIII:C obtained has a specific activity of 6 units/micrograms based on absorbance measurements. When analyzed on SDS gels, the unactivated VIII:C contains 3 bands of apparent molecular weight 166,000, 130,000 and 76,000. Thrombin treatment results in a 40 fold increase in activity and cleavage to products of 76,000, 67,000 an 50,000 and small amounts of lower molecular weight peptides. EDTA inactivation of the factor VIII:C results in the separation of the 166,000 and 130,000 chains from the 76,000 chain, suggesting a Ca++ dependent noncovalent interaction among the chains.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000941:Antigens / Q000302:isolation & purification*; D001202:Ascitic Fluid / Q000378:metabolism; D001666:Binding Sites, Antibody; D055598:Chemical Phenomena; D002621:Chemistry; D005169:Factor VIII / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D013552:Swine; D014841:von Willebrand Factor / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000941:Antigens; D014841:von Willebrand Factor; D005169:Factor VIII